Prokaryotic Promoters and Transcription

PCR Outline

• Qualitative PCR only determines if a sequence is present
• Quantitative PCR determines if a sequence is present and the amount
• Regular/Traditional PCR is qualitative
• Advantages: fast, easy, sensitive, can amplify small amounts from degraded/fixed samples
• Disadvantages: often requires prior knowledge of the sequence, short products, infidelity of replication
• Components needed for a PCR reaction and their roles:
  • Template DNA: blueprint for amplification
  • Primers: dictate where to amplify
  • DNA polymerase (Taq): synthesizes new strands, heat-resistant
  • Nucleotides (dNTPs): building blocks of DNA
• PCR cycle steps and temperatures:
  • Denaturation (95°C): separates DNA strands
  • Annealing (~55°C): primers bind to target sequence
  • Extension (72°C): new DNA synthesized

PCR Reaction

• Components needed for PCR reaction and their role:

  • Template DNA: The DNA sequence to be copied
  • Primers: Short DNA sequences that define the region to be amplified
  • DNA Polymerase: An enzyme that synthesizes new DNA strands by adding nucleotides to the 3' end of the primer. Taq polymerase is commonly used due to its heat stability.
  • Nucleotides (dNTPs): Deoxyribonucleoside triphosphates, the building blocks of DNA

• PCR cycle steps and temperatures:

  • Denaturation: Heating the DNA to a high temperature (typically 94-98°C) to separate the double-stranded DNA into single strands.
  • Annealing: Lowering the temperature (typically 50-65°C) to allow the primers to bind to their complementary sequences on the single-stranded DNA.
  • Extension: Raising the temperature again (typically 72°C) to allow the DNA polymerase to synthesize new DNA strands by extending the primers.

• Why Taq Polymerase is used:

  • Heat stable, can function at high temperatures required for PCR.

• Why PCR amplification is exponential

  • Each DNA molecule produced can be used as a template for further replication.

Primer Design

• Length: Usually 20 nucleotides
• Avoid these when designing primers:
  • Tandem repeats
  • Secondary structures (hairpins)
  • 3' end complementarity

Hot Start Methods

• Binding Protein Hot Start Method: Binding proteins bind to primers, then denaturation step inactivates them.
• Antibody Hot Start Method: Polymerase is bound by antibody preventing it from working until denaturation.

Genomic PCR vs. Reverse Transcriptase PCR (RT-PCR)

• Genomic PCR: Amplifies genomic DNA directly
• RT-PCR: Amplifies RNA by first converting it into cDNA

RT-PCR and cDNA

• RT-PCR converts RNA into cDNA using reverse transcriptase, then amplifies cDNA using PCR
• cDNA is single-stranded DNA produced from an RNA template using reverse transcriptase

qPCR

• Quantitative PCR
• Similar to regular PCR but measures the amplified DNA in real time
• Advantages: Monitor amplification real-time and accurate quantification of starting DNA material.
• Disadvantages: More expensive, requires more extensive instrumentation.

qPCR Data Analysis

• Ct value: Cycle number when fluorescence crosses a set threshold. Lower Ct values mean higher starting amounts of DNA target.
• Standard curve method: Use Ct to determine the starting DNA amount for unknown samples.
• AACt method: Used to compare Ct values of samples to a control (normalized)

Standard Curve

• Used to determine the initial starting amount of target template in experimental samples
• Slope measures reaction efficiency.
• Requires a dilution series of known template concentrations

Hot Start

• Method used to prevent non-specific amplification during PCR
• Keeps polymerase inactive until high temperatures are reached

Sequencing

• First Generation - Sanger Sequencing:
  • Based on chain termination, low cost, high accuracy
• Second Generation (NGS) - NGS:
  • High throughput, lower accuracy, cost-effective
• Third Generation - Long-Read Sequencing:
  • Very long reads, relatively lower accuracy but cost-effective for particular applications

Emulsion PCR

• Amplifies DNA molecules in micro-droplets.
• Ensures each droplet contains only one DNA molecule, increasing specificity of reaction.
• Useful for high-throughput applications, minimizes contamination

Droplet Digital PCR (ddPCR™)

• More sensitive than qPCR
• Distributes template DNA into numerous droplets
• Measures DNA in individual droplets to determine concentration with high accuracy.

Sanger (dideoxy) Sequencing

• DNA polymerase synthesizes new DNA strands
• Chain terminator nucleotides (ddNTPs) create different lengths, fragments read on a gel.

Cycle Sequencing

• PCR-based sequencing method
• Similar to Sanger, but uses fluorescent ddNTPs instead of radioactive ones.
• Fragments are separated based on fluorescence signal.

Other Sequencing Methods

• Ion Torrent PGM: • Measures hydrogen ion released when nucleotides are added to the growing DNA strand.
• Molecular Beacons, TaqMan Probes: • Use fluorescent probes to detect DNA synthesis.
• Oxford Nanopore: • DNA strands pass through a nanopore, changes in current are detected to determine nucleotide sequence.

Assembly

• Combining short DNA fragments (reads) into longer, contiguous sequences (contigs) with overlapping regions
• Challenges: repetitive sequences, errors in sequencing, and the need to use reference genes.

Prokaryotic Transcription and Control Outline

• Transcription: Making mRNA from DNA
• Translation: Making protein from mRNA
• Operon: cluster of genes transcribed as a single mRNA in prokaryotes
• Promoter: DNA region where RNA polymerase binds to initiate transcription
• Leader Sequences: Region in some operons preceding the transcribed protein-encoding sequence, important in transcriptional regulation.
• Repressors: Proteins that bind to DNA sequences (operator) to block RNA polymerase, hindering transcription.
• Activators: Proteins that bind to DNA to stimulate RNA polymerase activity.
• Attenuation: a secondary regulatory mechanism of some operons, in which termination of transcript occurs early in some conditions.
• Constitutive: Always transcribed with or without a regulatory protein
• Inducible: Transcribed only when needed based on the environmental signals for an induced response
• Repressible: Transcribed unless blocked by a regulatory protein

RNA Types & Structures

• mRNA (messenger RNA): codes for proteins
• tRNA (transfer RNA): carries amino acids to ribosomes
• rRNA (ribosomal RNA): forms part of ribosomes
• ncRNA (non-coding RNA): various functions, many of which are regulatory (e.g., sRNA).

This quiz explores key concepts related to prokaryotic promoters, including the functions of -10 and -35 sequences, the relationship between template strands and RNA transcripts, and factors influencing promoter strength. Test your understanding of transcription initiation and consensus sequences in this engaging quiz.